DNA repair provides a major defense mechanism against DNA lesions and their potential consequences, including mutagenesis, carcinogenesis, or cell death. The nucleotide excision repair (NER) pathway is a general repair process that removes a remarkably diverse array of structurally unrelated lesions, ranging from UV-induced photoproducts, chemical adducts, abasic sites to certain types of cross-links (Van Houten, Microbiol. Rev. 54, 18-51 (1990)).
The mechanism of NER is best studied in the bacterium Escherichia coli. This pathway, consisting of five steps: damage recognition, incision, excision, DNA repair synthesis and ligation, is error-free and leads to restoration of the integrity of the genetic information. The NER in bacterial cells is initiated by a combined action of three proteins, a UvrA protein, a UvrB protein, and a UvrC protein, leading to recognition and incision of damaged DNA. The three proteins, which are also referred to as UvrABC endouclease, are typically not stable. For instance, E. coli UvrA protein has been shown to be heat labile, especially in dilute concentrations with a t1/2 of less than five minutes at 37° C. (Zou et al., J. Biol. Chem. 273, 12887-12892 (1998)).
The UvrA protein, which has a moderate affinity for damaged DNA (Van Houten, Microbiol. Rev. 54, 18-51 (1990); Seeberg et al., Proc. Natl. Acad. Sci. USA 79, 988-992 (1982); Claassen et al., J. Biol. Chem. 266, 11388-11394 (1991)), associates with the UvrB protein to form a UvrA2UvrB complex that tracks along DNA (Koo et al., Proc. Natl. Acad. Sci. USA 88, 1212-1216 (1991)) and delivers UvrB to the damaged site. UvrA, in an ATP-dependent reaction, dissociates from this complex at the damaged site and a very stable UvrB-DNA complex is formed (Orren et al., Proc. Natl. Acad. Sci. USA 86, 5237-5241 (1989); Orren et al., J. Biol. Chem. 265, 15796-15803 (1990)). This complex constitutes a high affinity binding site for the UvrC protein, which upon binding to a UvrB-DNA complex, triggers incision at the 4th to the 7th phosphodiester bonds 3′ to the damaged site (Lin et al., J. Biol. Chem. 267, 17693-17700 (1992); Moolenaar et al., J. Biol. Chem. 270, 30508-30515 (1995)). Immediately after the 3′ incision, 5′ incision occurs at the 8th phosphate group 5′ to the DNA lesion (Lin et al. J. Biol. Chem. 267, 17688-17692 (1992); Zou et al., Biochemistry 34, 13582-13593 (1995)). Prokaryotic NER leads to the excision of lesions as oligomers 12-15 nucleotides in length.
Within this reaction cascade the UvrB protein plays a central role since it interacts with all the components of excision repair, namely UvrA, UvrC, UvrD (helicase 11), DNA polymerase I and DNA (Sancar and Sancar (1988) Annu. Rev. Biochem., 57, 29-67; Orren et al. (1992) J. Biol. Chem., 267, 780-788). Sequence comparisons have identified six helicase motifs throughout the sequence of UvrB (Gorbalenya et al. (1989) Nucleic Acids Res., 17, 4713-4730) indicating that UvrB is a member of the helicase II superfamily, like the helicases Rad3 and XPD involved in eukaryotic NER (Sung et al. (1987) Proc. Natl. Acad. Sci. USA, 84, 8951-8955; Sung et al. (1993) Nature, 365, 852-855). In complex with UvrA, UvrB has been shown to have helicase-like activity in a reaction requiring the hydrolysis of ATP (Oh and Grossman (1987) Proc. Natl Acad. Sci. USA, 84, 3638-3642; Oh and Grossman (1989) J. Biol. Chem., 264, 1336-1343). In addition to its possible role of tracking along the DNA, UvrB alters the affinity of the UvrA2B complex towards more bulky adducts compared with UvrA alone (Snowden and Van Houten (1991) J. Mol. Biol., 220, 19-33; Visse et al. (1991) J. Biol. Chem., 266, 7609-7617; Visse et al. (1994) Biochemistry, 33, 1804-1811). The UvrA dimer is sufficient in recognizing damaged DNA, but it is the UvrA2B complex that binds to damaged sites with increased specificity and allows efficient DNA damage recognition in vivo. Furthermore, this damage processing, which involves bending and unwinding of the DNA (Lin et al. (1992) J. Biol. Chem., 267, 17693-17700; Visse et al. (1994) Biochemistry, 33, 9881-9888; Zou and Van Houten (1999) EMBO J., 18, 4889-4901), leads to a stable UvrB-DNA pre-incision complex serving as a scaffold for the binding of UvrC.
Genetic and biochemical data show the prokaryotic pattern of NER to be present in more than 30 different eubacterial species, including three thermophilic microorganisms, Thermus thermophilus (Yamamoto et al., Gene 171, 103-106 (1996)), Aquifex aeolicus (Deckert et al., Nature 392, 353-358 (1998)), and Thermotoga maritima (Nelson et al., Nature 399, 323-329 (1999)). Sequence analyses indicate a high level of amino acid sequence similarity between Uvr proteins from different, even phylogenetically very distant bacterial species. Furthermore, it has been shown that the UvrA and UvrB proteins from E. coli, a gram-negative bacterium, can be complemented both in vitro and in vivo with the UvrC protein from gram-positive bacterium, Bacillus subtilis (Lin et al., J. Biol. Chem. 265, 21337-21341 (1990)) indicating a significant evolutionary conservation of the NER system among Eubacteria. More recently, homologues of uvrA, uvrB, and uvrC genes have been found in the genome of Methanococcus thermoautotrophicum (Smith et al., J. Bacteriol. 179, 7135-7155 (1997)), a member of the third kingdom of organisms, Archaea. In contrast, the genome sequences of archaeal Methanococcus janaschii (Bult et al., Science 273, 1058-1073 (1996)) and Archaeoglobus fulgidus (Klenk et al., Nature 310, 364-370 (1997)) do not contain uvr gene homologues, suggesting the presence of a novel pattern of NER pathway at least in some archaeal species.